Recently, regenerative therapy has attracted attention as a novel approach to severe organ failure or intractable diseases. Regenerative therapy is a combination of genetic engineering, cell tissue engineering, regenerative medicine, and the like. Many researchers over the world are vigorously working on this important and challenging subject of research in the 21-century advanced medical practice.
The scale of the market associated with regenerative medicine (tissue engineering) is estimated as about 500 billion US dollars in the world and about 50 billion US dollars in Japan according to the material prepared by the New Energy and industrial Technology Development Organization. Only tissue engineering products account for about 100 billion US dollars in the world. The regenerative medicine is greatly expected to create the next-generation industry.
The present inventors have made efforts to develop regenerative therapy in the field of musculoskeletal and cardiovascular tissues, and have reported a combination therapy of cell implantation and a growth factor administration, or a tissue implantation regeneration therapy based on tissue engineering. However, regenerative therapy based on cell or tissue implantation requires a source of autologous cells. A stable and abundant source of such cells is urgently required and important. A number of cells in musculoskeletal tissue have a high level of self-repairing ability. It has been reported that there is a stem cell among the cells of the musculoskeletal tissue.
It has been demonstrated that a cell derived from skeletal muscle (Jankowiski R. J., Huand J. et al, Gene Ther., 9:642-647, 2002), fat (Wickham M. Q. et al., Clin. Orthop., 2003, 412, 196-212), umbilical cord blood (Lee O. K. et al., Blood, 2004, 103:1669-75), tendon (Salingcarnboriboon R., Exp. Cell. Res., 287:289-300, 2002), bone marrow (Pitterger M. F. et al., Science, 284:143-147, 1999), and synovium (Arthritis Rheum. 2001 44:1928-42) is undifferentiated and has the potential to differentiate into various cells.
Conventionally, when cell therapy is performed for repair or regeneration of tissue, most research employs a biological scaffold to maintain the accumulation of cells, allow cells to grow, maintain pluripotency, protect cells from mechanical stress on a treated site, or the like. However, most scaffolds contain a biological (animal) material, a biomacromolecule material, or the like, of which influence on the safety of organism cannot be fully predicted.
A cell implanting method without a scaffold has been reported by Kushida A., Yamato M., Konno C., Kikuchi A., Sakurai Y., Okano T., J. Biomed. Mater. Res., 45:355-362, 1999, in which a cell sheet is produced using a temperature sensitive culture dish. Such a cell sheet engineering technique is internationally appraised due to its originality. However, a single sheet obtained by this technique is fragile. In order to obtain the strength that can withstand surgical manipulation, such as implantation, a plurality of sheets need to be assembled, for example.
When a nano-biointerface technology is used to fix a temperature responsive polymer (PIPAAm) onto a plastic mold, such as a Petri dish, for cell culture, the polymer surface is reversibly changed at 31° C. between hydrophilicity and hydrophobicity. Specifically, when the temperature is 31° C. or more, the surface of the Petri dish is hydrophobic so that cells or the like can adhere thereto. In this situation, the cells secrete extracellular matrix (ECM; for example, adhesion molecules which are proteins having a function like a “glue”) and adhere to the surface of the Petri dish, so that the cells can grow. See, Okano T., Yamada N., Sakai H., Sakurai Y., J. Biomed. Mater. Res., 1993, 27:1243-1251; Kushida A., Yamato M., Konno C., Kikuchi A., Sakurai Y., Okano T., J. Biomed. Mater. Res. 45:355-362, 1999; and Shimizu T., Yamato M., Akutsu T. et al., Circ. Res., 2002, Feb. 22; 90 (3):e40.
When the temperature is 31° C. or less, the surface of the Petri dish is hydrophilic. The cells which have adhered to the Petri dish are readily detached, though the cells still maintain adhesion molecules. This is because the surface of the Petri dish to which the cells have adhered no longer exists at 31° C. or less.
Even when such a Petri dish having a fixed temperature responsive polyer (e.g., tradename: UpCell and RepCell) is used to culture cells and detach the cells, an extracellular matrix is not appropriately provided. Thus, there has been no actually practical synthetic tissue developed. See, Okano T., Yamada N., Sakai H., Sakurai Y., J. Biomed. Mater. Res., 1993, 27:1243-1251; Kushida, A., Yamato M., Konno C., Kikuchi A., Sakurai Y., Okano T., J. Biomed. Mater. Res. 45:355-362, 1999; and Shimizu T., Yamato M., Akutsu T. et al., Circ. Res., 2002, Feb. 22; 90(3):e40.
WO00/51527 and WO03/024463 reported that cells are cultured on a semipermeable membrane using alginate gel. However, the resultant tissue is poorly integrated with an extracellular matrix and is not free of a scaffold. In addition, the cells in the tissue are not self organized. The tissue has no self-supporting ability. The cells no longer have a differentiation potential. The tissue loses morphological plasticity in terms of three-dimensional structure. Therefore, the tissue is not suitable for cell implantation.
Use of a scaffold is considered to be problematic in implantation therapy because of adverse side effects. Therefore, there is a demand for the advent of a scaffold-free technique.
Conventional methods for producing tissue sheets have the following drawbacks: it is not possible to produce a very large sized sheet; it is not possible to produce a sheet having biological integration in three dimensions; when a sheet is detached after sheet production, the sheet is broken into pieces; and the like.
Therefore, there is a keen demand for a synthetic tissue, which is developed by culture processes, capable of withstanding an implantation operation, capable of being used in an actual operation.
By conventional techniques, it is difficult to isolate a synthetic tissue from a culture base material after tissue culture, and it is substantially impossible to produce a large sized tissue piece. Therefore, conventional synthetic tissues, such as tissue sheets, cannot be used in medical application in view of size, structure, mechanical strength, and the like. It is difficult to develop a synthetic tissue using conventional techniques. Therefore, unfortunately their supplies are limited.
An object of the present invention is to provide a synthetic tissue produced by cell culture, which is feasible to implantation surgery.
Specifically, an object of the present invention is to provide a synthetic tissue having a three-dimensional structure and self-supporting ability, being free of a scaffold, and maintaining a differentiation potential if the tissue possesses it.
Still another object of the present invention is to provide a method and a pharmaceutical agent for treating an injury of a tissue or the like when a replacement or resurfacing therapy is required.